Reduced epidermal thickness,nerve degeneration and increased pain-related behavior in rats with diabetes type 1 and 2

To examine the mechanisms contributing to pain genesis in diabetic neuropathy, we investigated epidermal thickness and number of intraepidermal nerve fibers in rat foot pad of the animal model of diabetes type 1 and type 2 in relation to pain-related behavior. Male Sprague-Dawley rats were used. Diabetes type 1 was induced with intraperitoneal injection of streptozotocin (STZ) and diabetes type 2 was induced with a combination of STZ and high-fat diet. Control group for diabetes type 1 was fed with regular laboratory chow, while control group for diabetes type 2 received high-fat diet. Body weights and blood glucose levels were monitored to confirm induction of diabetes. Pain-related behavior was analyzed using thermal (hot, cold) and mechanical stimuli (von Frey fibers, number of hyperalgesic responses). Two months after induction of diabetes, glabrous skin samples from plantar surface of the both hind paws were collected. Epidermal thickness was evaluated with hematoxylin and eosin staining. Intraepidermal nerve fibers quantification was performed after staining skin with polyclonal antiserum against protein gene product 9.5. We found that induction of diabetes type 1 and type 2 causes significant epidermal thinning and loss of intraepidermal nerve fibers in a rat model, and both changes were more pronounced in diabetes type 1 model. Significant increase of pain-related behavior two months after induction of diabetes was observed only in a model of diabetes type 1. In conclusion, animal models of diabetes type 1 and diabetes type 2 could be used in pharmacological studies, where cutaneous changes could be used as outcome measures for predegenerative markers of neuropathies.     

Neurotrophic modulation of myelinated cutaneous innervation and mechanical sensory loss in diabetic mice

Human diabetic patients often lose touch and vibratory sensations, but to date, most studies on diabetes-induced sensory nerve degeneration have focused on epidermal C-fibers. Here, we explored the effects of diabetes on cutaneous myelinated fibers in relation to the behavioral responses to tactile stimuli from diabetic mice. Weekly behavioral testing began prior to streptozotocin (STZ) administration and continued until 8 weeks, at which time myelinated fiber innervation was examined in the footpad by immunohistochemistry using antiserum to neurofilament heavy chain (NF-H) and myelin basic protein (MBP). Diabetic mice developed reduced behavioral responses to non-noxious (monofilaments) and noxious (pinprick) stimuli. In addition, diabetic mice displayed a 50% reduction in NF-H-positive myelinated innervation of the dermal footpad compared with non-diabetic mice. To test whether two neurotrophins nerve growth factor (NGF) and/or neurotrophin-3 (NT-3) known to support myelinated cutaneous fibers could influence myelinated innervation, diabetic mice were treated intrathecally for 2 weeks with NGF, NT-3, NGF and NT-3. Neurotrophin-treated mice were then compared with diabetic mice treated with insulin for 2 weeks. NGF and insulin treatment both increased paw withdrawal to mechanical stimulation in diabetic mice, whereas NT-3 or a combination of NGF and NT-3 failed to alter paw withdrawal responses. Surprisingly, all treatments significantly increased myelinated innervation compared with control-treated diabetic mice, demonstrating that myelinated cutaneous fibers damaged by hyperglycemia respond to intrathecal administration of neurotrophins. Moreover, NT-3 treatment increased epidermal Merkel cell numbers associated with nerve fibers, consistent with increased numbers of NT-3-responsive slowly adapting A-fibers. These studies suggest that myelinated fiber loss may contribute as significantly as unmyelinated epidermal loss in diabetic neuropathy, and the contradiction between neurotrophin-induced increases in dermal innervation and behavior emphasizes the need for multiple approaches to accurately assess sensory improvements in diabetic neuropathy.     

Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy

Diabetes is the most prevalent metabolic disorder in the United States, and between 50% and 70% of diabetic patients suffer from diabetes-induced neuropathy. Yet our current knowledge of the functional changes in sensory nerves and their distal terminals caused by diabetes is limited. Here, we set out to investigate the functional and morphological consequences of diabetes on specific subtypes of cutaneous sensory nerves in mice. Diabetes was induced in C57Bl/6 mice by a single intraperitoneal injection of streptozotocin. After 6-8 wk, mice were characterized for behavioral sensitivity to mechanical and heat stimuli followed by analysis of sensory function using teased nerve fiber recordings and histological assessment of nerve fiber morphology. Diabetes produced severe functional impairment of C-fibers and rapidly adapting Aβ-fibers, leading to behavioral hyposensitivity to both mechanical and heat stimuli. Electron microscopy images showed that diabetic nerves have axoplasm with more concentrated organelles and frequent axon-myelin separations compared with control nerves. These changes were restricted to the distal nerve segments nearing their innervation territory. Furthermore, the relative proportion of Aβ-fibers was reduced in diabetic skin-nerve preparations compared with nondiabetic control mice. These data identify significant deficits in sensory nerve terminal function that are associated with distal fiber loss, morphological damage, and behavioral hyposensitivity in diabetic C57Bl/6 mice. These findings suggest that diabetes damages sensory nerves, leading to functional deficits in sensory signaling that underlie the loss of tactile acuity and pain sensation associated with insensate diabetic neuropathy.     

Dissociation of thermal hypoalgesia and epidermal denervation in streptozotocin-diabetic mice

The quantification of epidermal innervation, which consists primarily of heat-sensitive C-fibers, is emerging as a tool for diagnosing and staging diabetic neuropathy. However, the relationship between changes in heat sensitivity and changes in epidermal innervation has not yet been adequately explored. Therefore, we assessed epidermal nerve fiber density and thermal withdrawal latency in the hind paw of Swiss Webster mice after 2 and 4 weeks of streptozotocin-induced diabetes. Thermal hypoalgesia developed after only 2 weeks of diabetes, but a measurable reduction in PGP9.5-immunoreactive epidermal nerve fiber density did not appear until 4 weeks. These data suggest that impaired epidermal nociceptor function contributes to early diabetes-induced thermal hypoalgesia prior to the loss of peripheral terminals.     

Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: a multimodal analysis

One important complication of diabetes is damage to the peripheral nervous system. However, in spite of the number of studies on human and experimental diabetic neuropathy, the current therapeutic arsenal is meagre. Consequently, the search for substances to protect the nervous system from the degenerative effects of diabetes has high priority in biomedical research. Neuroactive steroids might be interesting since they have been recently identified as promising neuroprotective agents in several models of neurodegeneration. We have assessed whether chronic treatment with progesterone (P), dihydroprogesterone (DHP) or tetrahydroprogesterone (THP) had neuroprotective effects against streptozotocin (STZ)-induced diabetic neuropathy at the neurophysiological, functional, biochemical and neuropathological levels. Using gas chromatography coupled to mass-spectrometry, we found that three months of diabetes markedly lowered P plasma levels in male rats, and chronic treatment with P restored them, with protective effects on peripheral nerves. In the model of STZ-induced of diabetic neuropathy, chronic treatment for 1 month with P, or with its derivatives, DHP and THP, counteracted the impairment of nerve conduction velocity (NCV) and thermal threshold, restored skin innervation density, and improved Na(+),K(+)-ATPase activity and mRNA levels of myelin proteins, such as glycoprotein zero and peripheral myelin protein 22, suggesting that these neuroactive steroids, might be useful protective agents in diabetic neuropathy. Interestingly, different receptors seem to be involved in these effects. Thus, while the expression of myelin proteins and Na(+),K(+)-ATPase activity are only stimulated by P and DHP (i.e. two neuroactive steroids interacting with P receptor, PR), NCV, thermal nociceptive threshold and intra-epidermal nerve fiber (IENF) density are also affected by THP, which interacts with GABA-A receptor. Because, a therapeutic approach with specific synthetic receptor ligands could avoid the typical side effects of steroids, future experiments will be devoted to evaluating the role of PR and GABA-A receptor in these protective effects.     

Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques: An Alternate Approach to Investigate HIV-Induced PNS Damage

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings.Peripheral neuropathy (PN) is the most frequent neurological complication caused by HIV-1, affecting more than one-third of infected persons, including patients receiving combination antiretroviral therapy.^1,2 The typical clinical presentation is known as distal sensory polyneuropathy, a length-dependent neuropathy that is characterized by bilateral aching, painful numbness or burning, and is most pronounced in the lower extremities.^3,4 Although HIV-induced PN (HIV-PN) is not life threatening, this debilitating disorder greatly compromises patient quality of life.¹ Currently, skin biopsy is the accepted standard for measuring the loss of small, unmyelinated C fibers in the epidermis, one of the earliest detectable signs of damage to the peripheral nervous system (PNS).^5–7 However, skin biopsy is an invasive procedure, and longitudinal assessment requires repeated surgical biopsies. Electrophysiological testing to measure properties of peripheral nerve conduction is not considered a viable alternative because current methods lack the sensitivity required to detect damage to small, unmyelinated fibers, especially in early stages of disease.^8,9 For these reasons, new, sensitive, noninvasive methods of assessing small fiber nerve damage are urgently needed to detect and monitor PN in persons infected with HIV.Like HIV, small sensory nerve fiber loss is common in patients with diabetes mellitus and results in a clinical syndrome that closely resembles HIV distal sensory polyneuropathy.¹⁰ Of interest, studies have documented the utility of measuring changes in corneal sensory innervation to track diabetic neuropathy as an alternative to measuring epidermal nerve fiber (ENF) density via skin biopsy. In particular, decreases in the nerve density in the corneal subbasal plexus (SBP) have been reported in both isolated corneal whole mount studies and by noninvasive in vivo corneal confocal microscopy (CCM).^11–15Although corneal nerve assessments have found increasing value as a surrogate to evaluate ENFs in diabetic PN, the use of corneal alterations in tracking HIV-induced neuropathy has yet to be explored. To study the pathogenesis of HIV-induced PNS disease, our group developed a SIV-infected macaque model that closely recapitulates key PNS alterations seen in patients with HIV with PN. These changes include macrophage infiltration, SIV replication, and neuronal loss in sensory ganglia, including the trigeminal ganglia, which house the cell bodies of sensory neurons that innervate the cornea.^16,17 In this report, we aimed to determine whether SIV infection leads to decreases in corneal nerve fiber (CNF) density, whether changes in corneal nerves correspond with the extent of SIV replication and the severity of cellular immune responses in the trigeminal ganglia, and whether CNF density correlates with ENF length, thereby setting the stage for follow-up CCM investigation. To achieve optimal immunostaining of the corneal SBP, we modified an immunohistochemical staining method developed by Marfurt et al.¹⁸ Because conventional image analysis of the corneal nerve is labor intensive and requires trained observers, we developed two novel counting methods: a relatively simple manual counting method of nerve fibers and an automated method of nerve detection and counting to facilitate efficient, objective assessment of nerve fiber density.     

Diabetic neuropathy and nerve regeneration

Diabetic neuropathy is the most common peripheral neuropathy in western countries. Although every effort has been made to clarify the pathogenic mechanism of diabetic neuropathy, thereby devising its ideal therapeutic drugs, neither convinced hypotheses nor unequivocally effective drugs have been established. In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance nerve regeneration as well as prevent nerve degeneration. Nerve regeneration or sprouting in diabetes may occur not only in the nerve trunk but also in the dermis and around dorsal root ganglion neurons, thereby being implicated in the generation of pain sensation. Thus, inadequate nerve regeneration unequivocally contributes to the pathophysiologic mechanism of diabetic neuropathy. In this context, the research on nerve regeneration in diabetes should be more accelerated. Indeed, nerve regenerative capacity has been shown to be decreased in diabetic patients as well as in diabetic animals. Disturbed nerve regeneration in diabetes has been ascribed at least in part to all or some of decreased levels of neurotrophic factors, decreased expression of their receptors, altered cellular signal pathways and/or abnormal expression of cell adhesion molecules, although the mechanisms of their changes remain almost unclear. In addition to their steady-state changes in diabetes, nerve injury induces injury-specific changes in individual neurotrophic factors, their receptors and their intracellular signal pathways, which are closely linked with altered neuronal function, varying from neuronal survival and neurite extension/nerve regeneration to apoptosis. Although it is essential to clarify those changes for understanding the mechanism of disturbed nerve regeneration in diabetes, very few data are now available. Rationally accepted replacement therapy with neurotrophic factors has not provided any success in treating diabetic neuropathy. Aside from adverse effects of those factors, more rigorous consideration for their delivery system may be needed for any possible success. Although conventional therapeutic drugs like aldose reductase (AR) inhibitors and vasodilators have been shown to enhance nerve regeneration, their efficacy should be strictly evaluated with respect to nerve regenerative capacity. For this purpose, especially clinically, skin biopsy, by which cutaneous nerve pathology including nerve regeneration can be morphometrically evaluated, might be a safe and useful examination.     

The Impact of Type 1 Diabetes Mellitus on Corneal Epithelial Nerve Morphology and the Corneal Epithelium

Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis.Diabetes mellitus (DM) is a severe metabolic disease with increasing prevalence worldwide. Diabetic neuropathy is one of the most common complications in DM and increases in incidence with both age and duration of disease.¹ At the ocular surface, loss of corneal innervation in DM is associated with reduced corneal sensitivity and altered tear secretion.^2–4 Reduced aqueous tear production from decreased lacrimal gland innervation and an abnormal blink reflex contribute to the high incidence of dry eye that frequently is encountered clinically. In addition, loss of trophic support, which is essential for homeostasis of the epithelium, is believed to contribute to epithelial fragility, corneal erosions, and persistent epithelial defects.⁵ Changes in corneal thickness also have been reported. In the earlier stages of disease, central corneal thickening occurs as a result of endothelial dysfunction and increased corneal edema.^6,7 Later stages of disease, however, result in thinning of the corneal epithelium, which is associated with a more severe neurotrophic state.⁸ Restoration of corneal epithelial homeostasis and the associated corneal nerve plexus presents an important clinical challenge because many of these neurotrophic conditions are highly refractory to conventional therapies.In vivo confocal microscopy (IVCM) is used increasingly in clinical studies to evaluate disease-related changes in the subbasal nerve plexus (SBNP). In diabetes, morphologic alterations in the SBNP have been associated with both retinopathy and peripheral neuropathy.^9–12 Although rapid and noninvasive, the use of confocal microscopy in clinical studies prohibits examination of the terminal epithelial nerves (TENs) and raises many questions regarding whether the loss documented by the confocal examination represents one or more fibers, and the type of fibers affected.¹³ To address this gap, immunohistochemical studies using rodent models of diabetes have investigated the effects of hyperglycemia on the SBNP and TENs. By using two-dimensional imaging techniques, these studies have reported reductions in both the SBNP and TENs, with early loss of TENs preceding SBNP damage.¹⁴ However, because of the tortuosity and complex branching patterns of the TENs as they run throughout the corneal epithelium, analysis of TENs from two-dimensional data sets limit the data that can be extracted and therefore requires more advanced three-dimensional (3D) modeling. Moreover, none of the rodent studies to date have shown a detectable effect on central corneal epithelial thickness or basal epithelial cell density (BECD), as seen in humans, as a consequence of disease state or nerve loss.^14,15Here, we investigated the effects of type 1 DM on the mouse corneal epithelium. Our primary objective was to evaluate the effects of type 1 DM on total corneal and sublayer thickness and BECD.^16,17 Our secondary objective was to assess the corresponding effects on the neural architecture using 3D volumetric reconstruction of the SBNP and TENs in situ. This allowed for a systematic evaluation of corneal nerve thickness and cellular changes in response to disease duration and animal age.     

Skin biopsy as a beneficial procedure for morphological evaluation of diabetic neuropathy

In an attempt to evaluate the morphological abnormalities of dermal non-myelinated nerve fibers of diabetics and elucidate the pathogenesis of diabetic peripheral neuropathy, the terminal part of peripheral nerve in the upper dermis was observed on electron microscopy using skin samples biopsied in 10 diabetics with symptomatic neuropathy and 6 age-matched controls. In diabetics, the density of nerve fibers was significantly lower than in controls. In addition, swelling, lytic change and vacuolation in the axon, multiplication of basement membrane of the Schwann cell and Schwann cell cluster devoid of axon were more frequently observed in diabetics. The Schwann cell did not show significant structural alterations. These findings suggest that the axon is primarily involved, at least in the terminal region of nerve fiber, in diabetic peripheral neuropathy. It is also concluded that the skin biopsy technique is harmless, cosmetically not troublesome and might be beneficial for studying peripheral neuropathies including diabetic neuropathy.     

Effects of streptozotocin-induced diabetes on taste buds in rat vallate papillae

Some studies have documented taste changes in patients with diabetes mellitus (DM). In order to understand the relationships between taste disorders caused by DM and the innervation and morphologic changes in the taste buds, we studied the vallate papillae and their taste buds in rats with DM. DM was induced in these rats with streptozotocin (STZ), which causes the death of beta cells of the pancreas. The rats were sacrificed and the vallate papillae were dissected for morphometric and quantitative immunohistochemical analyses. The innervations of the vallate papillae and taste buds in diabetic and control rats were detected using immunohistochemistry employing antibodies directed against protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). The results showed that PGP 9.5- and CGRP-immunoreactive nerve fibers in the trench wall of diabetic vallate papillae, as well as taste cells in the taste buds, gradually decreased both intragemmally and intergemmally. The morphometry revealed no significant difference in papilla size between the control and diabetic groups, but there were fewer taste buds per papilla (per animal). The quantification of innervation in taste buds of the diabetic rats supported the visual assessment of immunohistochemical labeling, that the innervation of taste cells was significantly reduced in diabetic animals. These findings suggest that taste impairment in diabetic subjects may be caused by neuropathy defects and/or morphological changes in the taste buds.     

SKIN BIOPSY AS A BENEFICIAL PROCEDURE FOR MORPHOLOGICAL EVALUATION OF DIABETIC NEUROPATHY

In an attempt to evaluate the morphological abnormalities of dermal non-myelinated nerve fibers of diabetics and elucidate the pathogenesis of diabetic peripheral neuropathy, the terminal part of peripheral nerve in the upper dermis was observed on electron microscopy using skin samples biopsied in 10 diabetics with symptomatic neuropathy and 6 age-matched controls. In diabetics, the density of nerve fibers was significantly lower than in controls. In addition, swelling, lytic change and vacuolation in the axon, multiplication of basement membrane of the Schwann cell and Schwann cell cluster devoid of axon were more frequently observed in diabetics. The Schwann cell did not show significant structural alterations. These findings suggest that the axon is primarily involved, at least in the terminal region of nerve fiber, in diabetic peripheral neuropathy. It is also concluded that the skin biopsy technique is unharmful, cosmetically not troublesome and might be beneficial for studying peripheral neuropathies including diabetic neuropathy.     

The cutaneous silent period in diabetes mellitus

The cutaneous silent period (CSP) may be useful as a method for the evaluation of smaller and unmyelinated fiber dysfunctions. CSP refers to the brief interruption in voluntary contraction that follows strong electrical stimulation of a cutaneous nerve. The aim the present study is to establish whether CSP can be instrumental in the determination of diabetic neuropathy. The nerve conduction studies and CSP evaluations were both used in patients with Diabetes Mellitus and control group. All patients were given clinical neurological examinations for the determination of small-fiber neuropathy (SFN). The CSP values for patients with SFN were compared with values of those without SFN. The nerve conduction velocities had changed unfavorably in diabetic patients. No median nerve CSP reponse could be obtained in two of the diabetic patients. CSP latency (84.6+/-14.0) in diabetics was longer than controls (76.2+/-13.1) (p=0.018). The duration of CSP was similar for the two groups (p=0.46). The CSP latency showed a correlation with routine nerve conduction studies. While the CSP latencies (86.7+/-15.8) of patients who were clinically diagnosed with SFN were similar to the latencies (81.3+/-10.4) of patients without SFN (p=0.606), the duration of CSP (44.6+/-13.7) in patients with SFN was shorter than the duration (55.3+/-12.2) in patients without SFN (p=0.012). These results indicate that even though the CSP does not provide any advantage over routine electrodiagnostic studies in determining diabetic neuropathy, still it may be a useful method for the early detection of diabetic SFN.     

The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production

Diabetes related peripheral neuropathy involves both somatic and autonomic nerves and leads to an array of debilitating abnormalities. Mechanisms may include decreased neuronal conductance, reactive oxygen species, and decreased performance of the perineurium blood-nerve barrier. Here we studied the perineurium characteristics of the dorsal penile nerve in a rat model of diabetes related peripheral neuropathy. Immunohistochemistry showed extensive and perineurial cell-specific nitric oxide synthase2 staining in diabetic animals as compared to age matched controls (P<0.05); however no apparent difference in immunostaining pattern was observed for 3-nitrotyrosine (a stable biomarker of peroxynitrite formation). Significant reductions in connexins 32 and 26 were seen in the diabetic perineurium with no detectable levels of connexin 43 in either control or diabetic dorsal nerve. These data provide new evidence of perineurial cell inflammatory responses and altered gap junction protein expression during diabetes related neuropathies and suggests that strategies to protect this cell type may have therapeutic value.     

Evaluation of PMI-5011, an ethanolic extract of Artemisia dracunculus L., on peripheral neuropathy in streptozotocin-diabetic mice

We previously reported that PMI-5011, an ethanolic extract of Artemisia dracunculus L., alleviates peripheral neuropathy in high fat diet-fed mice, a model of prediabetes and obesity developing oxidative stress and pro-inflammatory changes in the peripheral nervous system. This study evaluated PMI-5011 on established functional, structural, and biochemical changes associated with Type I diabetic peripheral neuropathy. C57Bl6/J mice with streptozotocin-induced diabetes of a 12-week duration, developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia, and intra-epidermal nerve fiber loss. PMI-5011 (500?mg/kg/day for 7?weeks) alleviated diabetes-induced nerve conduction slowing, small sensory nerve fiber dysfunction, and increased intra-epidermal nerve fiber density. PMI-5011 blunted sciatic nerve and spinal cord 12/15-lipoxygenase activation and oxidative-nitrosative stress, without ameliorating hyperglycemia or reducing sciatic nerve sorbitol pathway intermediate accumulation. In conclusion, PMI-5011, a safe and non-toxic botanical extract, may find use in the treatment of diabetic peripheral neuropathy.     

Cutaneous nerves in atopic dermatitis

Although pruritus is the cardinal symptom of atopic dermatitis, its mechanism is not well understood. Free nerve endings in the skin are involved in pruritus as itching receptors. We studied the cutaneous nerve fibres in lichenified lesions of 16 patients with adult atopic dermatitis. On immunohistochemistry, fibres immunoreactive for neurofilament, neuron-specific enolase, and protein gene product 9.5 were observed in the papillary dermis and dermoepidermal junctions as well as in the epidermis. In these areas, no fibres stained positively for substance P, neuropeptide Y, vasoactive intestinal peptide, beta endorphin, somatostatin or serotonin. On electron microscopy, the ultrastructure of subepidermal and intraepidermal free nerve endings appeared to be essentially normal. However, the distribution density of the cutaneous nerve fibres was much higher than in normal controls, and the diameter of these fibres was much larger, because of the large number of axons in each nerve fibre. Degranulation of mast cells was not seen. These findings suggest that pruritus in lichenified atopic skin is probably not caused by damage to the cutaneous free nerve endings. In such lesions, the number of the cutaneous free nerve endings is greatly increased, but they may have a normal function.     

Peripheral neuropathy in patients with diabetes mellitus presenting as Bell's palsy

The aim of this study is to evaluate the peripheral nerves in diabetes mellitus with or without peripheral facial paralysis (PFP). A total of 49 diabetic patients with PFP within the last year (23 females, mean age 60.3 +/- 9.3), and 83 diabetic patients without PFP (41 females, mean age 59.5 +/- 9.9) were enrolled. The neurological examination, eye-blinking response, needle EMG and electrophysiological parameters of peripheral nerves were evaluated. The neuropathic pain, other positive and negative sensory symptoms were statistically more frequent in controls than the PFP group, while no difference was noted in total neuropathy score. Sural sensorial nerve action potential amplitudes were same in both groups, but median nerve amplitudes were significantly lower in the PFP group. It is suggested that PFP is not a part of multifocal neuropathy in diabetes mellitus. However, at least some parts of the nerve conduction studies were involved, focal neuropathies were more frequent while sensory neuropathies with small nerve fiber involvement were less frequent in diabetes patients with PFP.     

Early increase precedes a depletion of VIP and PGP-9.5 in the skin of insulin-dependent diabetics—correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18–46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n=6), with diabetes for 6 months to 3 years; group 2 (n=5), with the disease for 5–10 years; and group 3 (n=7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P <0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P <0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (>95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (<5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P=0·002), presence of clinical autonomic neuropathy (Fisher exact test P=0.04), and a reduced sural nerve conduction velocity (Fisher exact test P=0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.     

Sensory Innervation of the Female Human Umbilical Skin: Morphological Studies

Background: Sensory stimuli are conducted by several cutaneous sensory nerves and tactile corpuscles. The latter are specialized sensory organs that represent the starting point of many afferent sensory pathways. To date, our knowledge about the distribution of the sensory innervation in the umbilical skin of females is incomplete.

Aim of the study: To elucidate the morphology of the cutaneous innervation of the normal female umbilical skin.

Materials and methods: Biopsies of normal umbilical skin were obtained from female patients undergoing umbilical hernial repair. The specimens were processed for both immunohistological (antibodies against PGP9.5, pan-neuronal marker, and S-100 protein, marker of Schwann cells) and ultrastructural (transmission electron microscopy) examinations.

Results: The authors found abundant genital end-bulb-like structures, numerous epidermal and dermal Merkel cells, Meissner and Ruffini corpuscles, intraepidermal nerve terminals, and multiple free nerve endings surrounding the ducts and acini of the sweat glands.

Conclusions: The umbilical skin of females has abundant sensory innervation similar to that of the glans penis.     

Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes

Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.